PROJECT SUMMARY Hepatocellular carcinoma (HCC) is the fastest growing cause of cancer-related death in the United States and one of the leading causes of death in patients with cirrhosis ? the end result of any chronic liver injury. HCC is difficult to treat and outcomes have barely improved over the last 30 years, with 5-year survival under 20% and an incidence-to-mortality ratio near 1. Innovative strategies for detection, prevention, and treatment for HCC are desperately needed, the development of which will depend heavily on mouse models of liver cancer. The current models are inadequate because they do not take into account the cells of origin, which will likely have a significant impact on tumor initiation, maintenance, and therapeutic vulnerabilities. While the liver is appreciated for being a central metabolic hub and for its astounding regenerative capacity, cellular heterogeneity in the liver is mostly unexplored. Along the portal to central axis within the hepatic lobule, profound differences in gene expression, metabolism, hypoxia, and ploidy are observed. Whether or not these differences reflect differences in neoplastic potential, and whether or not they influence metabolic disease or carcinogenesis, is unclear. In recent years, there has been intense controversy about whether or not there is a liver stem/progenitor cell. To compound these debates, the injury assays used to drive HCC development often do not model common etiologies such as non- alcoholic steatohepatitis (NASH), which is emerging as the most common cause of cirrhosis in the U.S. We posit that a critical problem for HCC modeling is a lack of understanding of how different cell types contribute to cancer in the context of clinically relevant injuries. We believe that identifying the specific cellular subtypes that give rise to HCC and then being able to genetically perturb these cells is critical for us to better model HCC. Unfortunately, the field does not have mouse reagents to manipulate much of the heterogeneity in the liver. To address this, my lab has optimized CRISPR genome-editing methods to rapidly generate lineage tracing mice. Nine new CreER knock-in models that label different zone-specific and progenitor populations have been produced, effectively quadrupling the number of CreER lines available to our field. We will use these tools to trace and genetically manipulate cell types in a systematic fashion in order to identify the most important regenerative cell populations (Aim 1) and the HCC cell(s) of origin (Aim 2) in the context of clinically-relevant chemical and nutritional injuries. We will then ask if cell type specific gene manipulation of common HCC driver genes will help to uncover different transformation competencies between hepatocyte subpopulations (Aim 3). Success in this project will provide the community with a large panel of important CreER tools, allow the field to focus on important subpopulations that are more likely to transform, and reveal pathways that control tumor development in these cells.